A genogeographic study of the Kyrgyz mountain merino via microsatellite markers

The aim was to ascertain the genetic and geographical structure of the Kyrgyz mountain merino (KMM). We analyzed DNA samples of 109 Kyrgyz mountain merino specimens, bred in three state breeding factories (STB), including “Orgochor” in the Issykul Province, “Katta-Taldyk” in the Osh Province and STb named after Luschikhin in the Talas Province. We identified 126 alleles in 12 microsatellite markers (McM042, INRA006, McM527, ETH152, CSRD247, OarFCB20, INRA172, INRA063, MAF065, MAF214, INRA005, INRA023). There were 6 to 16 alleles in each locus (mean 10.500 ± 0.957 alleles per locus). We identified 67 rare alleles (prevalence less than 5.0 %), which made up 53.2 % of all alleles found. The greatest number of rare alleles was found in STR-markers of CSRD247, INRA023, INRA005, INRA006, MAF214 and OarFCB20. For each group, there were individual differences in the distribution of allele frequencies across all the STR loci studied. The most significant of them were as follows: with regard to the McM042 locus, allele 87 was major in the TALAS and OSH groups (35.6 and 45.7 %, respectively), whereas allele 95 was major in the ISSYK- KUL group (36.2 %); allele 154 was major in all groups with regard to the INRA172 locus, but it was 1.25 times less prevalent in the ISSYK-KUL and 1.66 times less prevalent in the OSH groups compared to TALAS (55.2 and 41.4 %, respectively), whereas alleles 156 and 158 were found only in the ISSYK-KUL group. Considering the ETH152 locus, 186 allele prevalence in the TALAS group was 51.1 %, but allele 190 was also markedly prevalent in the ISSYK-KUL and OSH groups, 34.5 and 34.3 %, respectively. The genetic division of the studied groups of KMM (with K from 3 to 10) was homogeneous – the contribution of each subcluster was equivalent. The AMOVA analysis revealed that the groups are located equidistantly. To conclude, the genetic diversity of the Kyrgyz mountain merino in three state breeding factories of the Kyrgyz Republic was high and comparable with each other.


Introduction
The sheep breeds of Kyrgyz mountain merino (KMM) are com moninallregionsoftheKyrgyzRepublic,whichdiffer in natural and climatic conditions. In order to improve the breeding and productive qualities of KMM sheep, intra-breed (zonal) types of sheep were created (Bekturov et al., 2017).
The Kyrgyz mountain merino was created in 1990-2006 on thebasis of theKyrgyz fine-wool breedusing sheep of the Australian merino breed and approved in 2006. The genetic structure of the breed includes 5 factory types and 24 factory lines. KMM sheep wool has high technological properties and has attributed to the highest quality categories ofmerinofinewool.Thesheeparealsoknownforhighmeat properties.
To assess the condition and preserve the features of the KMM gene pool, genogeographic studies are needed. The preservation and further improvement of the breed should be controlled by the genetic dynamics studies both in the breed as a whole and in the main breeding farms engaged in KMM breeding. We have previously shown that local breeds of farm animals (in particular, the Kyrgyz horse) are characterized withahighgeneticdiversity,butlocaldifferentiationisalso present,andthedifferencesaresignificantforanumberof high-altitude experimental zones (Isakova et al., 2021). In this regard, studies of similar structure are needed.
The information obtained during the molecular genetic analysis will complement the morphometric characteristics of breeding rams, repair rams and ewes, which will allow breeders to develop new and modify existing selection algorithms and schemes to maintain the inbreed KMM genetic diversity, as well as preserve the genetic identity of this breed. In the future, they plan a number of measures to improve the breeding qualities of KMM breed sheep.
Thus, the purpose of this study was to conduct a genogeographic study of the Kyrgyz mountain merino sheep breed.
Genotyping was carried out using a set of COrDIS Sheep (LLC "GORDIZ", Russia) reagents for multiplex analysis according to the manufacturer's recommendations. To correctly determine the genotype in the studied animals (amplicon size in bp), a sample with a control genotype included in the COrDIS Sheep kit was used. PCR were analyzed by capillary high-resolution electrophoresis using an automatic genetic analyzer Applied Biosystems 3500 (ThermoFisher, USA).
GenAIEx v. 6.503 was used to calculated the average number of alleles per locus (N a ),theeffectivenumberofalleles (N e ), the levels of expected (H e ) and observed (H o ) heterozygosity and the F IS coefficient(Excoffier,1991).STRUC TURE v. 2.3.4 allowed to calculate the Q criterion, which attributed each individual animal to the corresponding cluster (Pritchard et al., 2000). PPHELPER v. 1.0.10 web application (Francis, 2016) was used for graphical interpretation of the results obtained in STRUCTURE v. 2.3.4.
We used GenAlEx 6.503 software (Peakall et al., 2012) to analyze population genetic parameters, the degree of gene-ticdifferentiationbasedonmatricesofpairwiseF ST values, followed by visualization in Past v. 4.03 (Hammer et al., 2001).
The genetic structure of the studied samples of the KMM sheep breed was evaluated using principal component analysis (PCA) via clustering in STRUCTURE v. 2.3.4 (Pritchard et al., 2000) using a mixed model (the number of assumed K clusters from 3 to 10; the length of the burn-in period 50K; the Markov chain model Monte Carlo 5K). Ten iterations were completed for each K value. We also determined the A genogeographic study of the Kyrgyz mountain merino via microsatellite markers  opti malnumberofclusters(ΔK)inPOPHELPERv.1.0.10 web application, using the method proposed in (Evanno et al., 2005). All applicable international, national and/or institutional principles for the care and use of animals have been observed.

Results and discussion
The modern KMM sheep breed demonstrated a high level of inbreeding genetic variability, when 126 alleles were identi-fiedinthe12microsatellitemarkersstudied.Thenumberof alleles in each locus varied from 6 to 16 (mean 10.500 ± 0.957). Sixty-seven rare alleles (with a prevalence less than 5.0 %) wereidentified,53.2%ofthetotalnumberofidentifiedalleles. The greatest number of rare alleles was found for the STR markers CSRD247, INRA023,INRA005,INRA006,MAF214 and OarFCB20. In order to analyze KMM inbreeding genetic subdivision bred in three geographically isolated zones, we computed N a , N e , H o , H e , I values and the F IS coefficient, shown in Table 1.
The mean number of alleles per N a locus varied from 8.000 to 8.500 (mean 8.306 ± 2.595), whereas the maximum value was noted in the TALAS group from the M.N. Lushchikhin SBF.ThenumberofeffectiveN e alleles was the highest in the OSH sample from the Katta-Taldyk SBF. Shannon index, reflectingthecomplexityofthecommunitystructure, averaged 1.657 ± 0.333 with the highest value in the OSH sample from the Katta-Taldyk SBF. The observed heterozygosity H o as an indicator of the variability (polymorphism) of the populationreflectingtheproportionofheterozygousgenotypesin the experiment ranged from 0.693 to 0.764. The expected heterozygosity of H e as an indicator of the proportion of heterozygous genotypes, expected in the Hardy-Weinberg equilibrium, ranged from 0.730 to 0.770. Maximum values of H o and H e were in OSH from the Katta-Taldyk SBF. The mean value of F IS index was the most neutral (0.006) in this group and indicated a balanced prevalence of heterogeneous genotypes, i. e. the level of related mating of individuals in thesubpopulationwastheleastsignificantcomparedtothe remaining two groups. In general, when comparing N a , N e , H o , H e , I and the F IS coefficient, we found no statistically significantdifferencesbetweenthreestudiedsamplesasof the Student's t-test.
To assess the genetic subdivision of the KMM samples using STRUCTURE v. 2.3.4, we computed the Q criterion, whichcharacterizedthestratificationofeachindividualanimal in the corresponding group. A Q value of 75 % or higher confirmstheindividual'sattributiontoitscluster. Fig.2graphically demonstrates (using the PPHELPER v. 1.0.10 web application (http://pophelper.com/)) the results of the analysis carried out in STRUCTURE v. 2.3.4 (automatic sorting was carried out based on the attribution of a particular sample to a major cluster).
The genetic material of KMM sheep from three geographically isolated zones was used in the study (see Fig. 1). For all samples within clusters K = (3-10), there is a general uniformity of structure, whereas the contribution of each subcluster is equivalent. A pairwise comparison of the mean values of Q for three samples at K = 2 using analysis of variance showed no statisticallysignificantdifferences.Thus,F=0.112,p = 0.739 was for the pair TALAS/ISSYK-KUL; F = 0.023, p = 0.881, for the pair ISSYK-KUL/OSH; and F = 0.267, p = 0.607 was for the pair TALAS/OSH. This may result from the fact that the KMM subpopulations studied have common ancestors (for example, sheep producers); however, other factors may alsohaveaneffect.
Based on the analysis of F ST genetic distances calculated using the AMOVA algorithm for 12 STR markers, a PCR graph ГЕНЕТИКА ЖИВОТНЫХ / ANIMAL GENETICS wasconstructedreflectingthemutualsimilarity/differenceof the studied samples (Fig. 3). The information presented in Fig. 2 and 3 allows to conclude thatthestudiedsamplesofKMMdidnotdiffersignificantly from each other. However, each sample had features that arose fromthedifferencesintheallele'sprevalenceinthestudied STR loci, as well as the presence of rare and private (found only in one of the studied groups) alleles (Tables 2 and 3,  respectively).
Ingeneral,wefoundindividualdifferencesinthedistribu-tionprofileofallelefrequenciesacrossallthestudiedSTR lociforeachgroup.Themostsignificantofthosewereallele 87 in the major state in the McM042 locus (35.6 and 45.7 %, respectively) in the TALAS and OSH groups, whereas allele 95 was most prevalent (36.2 %) in the group ISSYK-KUL; major allele 154 for the INRA172 locus in all groups, however, in comparison with the TALAS group, its prevalence was 1.25 (ISSYK-KUL) and 1.66 (OSH) times lower, 55.2 and 41.4 %, respectively, and alleles 156 and 158 were found only in the ISSYK-KUL group; the prevalence of 186 allele in the ETH152 locus in the TALAS group was 51.1 %, whereas 190 allele was highly prevalent in ISSYK-KUL and OSH, 34.5 and 34.3 %, respectively.
We found that the mean N a in KMM (in the context of the STR markers studied in this paper) was the maximum in comparison with other studies. The calculated H o index also turned out to be one of the largest and was comparable with the values obtained for the breeds Wielkopolskaya (Poland), Olkuska (Poland), Kail (Pakistan) and Kazakh fine-haired (Kazakhstan) (Ahmed et al., 2014;Szumiec et al., 2018;Dossybayev et al., 2019). The high rates of KMM genetic diversity are directly related to the multi-stage breeding processes that thisbreedunderwentduringthelateXX-earlyXXIcentury.

Conclusion
Taken together, the genetic diversity of KMM breed sheep of the three state breeding plants of the Kyrgyz Republic is quite high and comparable to each other. We found it impossibletosingleoutagroupforwhichaqualitativelydifferent (highorlow)geneticdiversitywouldbedifferentcompared the other two groups.
Nevertheless, it cannot be denied that for Kyrgyz mountain merino sheep from the M.N. Lushchikhin SBF, there was still a slight shift towards inbreeding processes -F IS = = 0.052 ± 0.025 (the maximum individual values of this indicator were found for STR markers of INRA023 -0.120, . In this regard we assume that the positive shift of these markers (lack of heterozygotes) occurred due to the purposeful selection of individuals according to the economically valuable characteristics of wool, i. e. resulted from the association of these STR markers with the loci of quantitative traits QTL. However, such a relationship can only be assessed in further studies.